LPS-induced Disseminated Intravascular Coagulation Research Articles

Recombinant human erythropoietin attenuates hepatic dysfunction by suppressing hepatocellular apoptosis in lipopolysaccharide-induced disseminated intravascular coagulation in rats.

The aim of the present study was to clarify the effect of recombinant human erythropoietin (EPO) and low molecular weight heparin (LMWH) on a rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC). Experimental DIC was induced by sustained infusion of 5 mg/kg LPS for 4 h. EPO or LMWH was then administered to the LPS-induced DIC model. LPS-induced consumption coagulopathy, hemostatic activation and plasma TNF elevation remained unaltered in the LPS+EPO group, except for the D-dimer levels, and these abnormalities were significantly improved in the LPS+LMWH group. Plasma alanine aminotransferase (ALT) levels were markedly reduced in the LPS+EPO group, accompanied by a significant suppression of hepatocellular apoptosis. In the LPS+LMWH group, plasma creatinine levels and glomerular fibrin deposition were significantly attenuated, along with plasma ALT levels and hepatocellular apoptosis. Thus, a single administration of EPO may improve hepatic dysfunction by primarily exerting an anti-apoptotic, not anticoagulant, effect in the LPS-induced DIC model.

Modulating the rate of fibrin formation and clot structure attenuates microvascular thrombosis in systemic inflammation

Systemic inflammation can lead to coagulopathy and disseminated intravascular coagulation (DIC). In prior studies, the recombinant A2 domain of human von Willebrand factor (VWF; A2 protein) attenuated DIC and decreased mortality in lipopolysaccharide (LPS)-treated mice. Here, we performed studies to dissect the mechanism by which the A2 protein moderates DIC. We used confocal microscopy to analyze the fibrin clot structure in plasma from healthy humans and endotoxemic mice, turbidity assays to examine fibrin polymerization, and a murine model for LPS-induced DIC and introduced a loss-of-function mutation into the A2 protein for fibrin. The mutation of the residue E1567 located in the α2 helix of the folded A2 domain of VWF inhibited binding activity for fibrin, possibly mapping a novel region containing a putative binding site for fibrin. The A2 protein increased the initial rate of change of fibrin polymerization, intercalated into the fibrin network, and modified the resultant clot structure in vitro. Furthermore, ex vivo experiments using plasma from mice with endotoxemia treated with the A2 protein revealed an increased rate of fibrin formation and an altered clot structure as compared with plasma from nontreated sick animals. Moreover, and in contrast to the A2 mutant, the A2 protein improved survival and reduced fibrin deposition and microvascular thrombosis in mice with endotoxemia-induced DIC. Importantly, in vivo and in vitro studies indicated that the A2 protein did not affect experimental thrombosis. Thus, we provide evidence for a novel treatment to attenuate systemic inflammation-induced coagulopathy/DIC via targeting fibrin formation, without an increased risk for bleeding.

Paeoniflorin alleviates lipopolysaccharide-induced disseminated intravascular coagulation by inhibiting inflammation and coagulation activation.

Lipopolysaccharide (LPS) is a toxic component of the outer membrane of gram-negative bacteria that can activate the blood coagulation system, leading to disseminated intravascular coagulation (DIC). DIC is a syndrome characterized by thromboembolism and multiple organ failure. Herein, the beneficial effect of paeoniflorin (PF) on the alleviation of LPS-induced DIC was investigated with an experimental DIC mouse model. Briefly, mice were randomly divided into the following six groups: (1) control; (2) LPS; (3) heparin; (4) low-PF treatment; (5) medium-PF treatment; and (6) high-PF treatment. The histological morphology of the liver and kidney was observed, and the coagulation indicators (such as prothrombin time), function indicators (such as alanine transferase), and inflammatory factors (such as TNF-α) were detected. Additionally, an in vitro cell inflammation model using RAW 264.7 murine macrophages was established. Activation of the nuclear factor kappa B (NF-κB) signaling pathway and tumor necrosis factor-α (TNF-α) were determined by western blotting. Based on our findings, PF could significantly improve the histological morphology of the liver and kidney, indicating that PF protects the liver and kidney against damage induced by LPS. Additionally, PF improved the function and coagulation indicators and reduced the production of inflammatory factors. In vitro, PF inhibited the expression of TNF-α by suppressing NF-κB signaling pathway activation. Collectively, our findings support the hypothesis that PF has anti-inflammatory and anticoagulation effects for the alleviation of LPS-induced DIC. PF is thus a potential co-treatment option for DIC.

Tetrahydropalmatine has a therapeutic effect in a lipopolysaccharide-induced disseminated intravascular coagulation model.

ObjectivesThe aim of this study was to determine the therapeutic effects of tetrahydropalmatine (Tet) on disseminated intravascular coagulation (DIC) by exploring the role of Tet using a lipopolysaccharide (LPS)-induced DIC model.Methods/Materials: We established a mouse DIC model by injecting LPS. Hematoxylin-eosin (HE) staining was performed to detect liver and kidney damage. Blood samples were obtained to determine liver and kidney injury indexes, coagulation indexes, and inflammatory cytokines. An in vitro cell inflammation model was also established. Tumor necrosis factor-α (TNF-α) levels and nuclear factor kappa B (NF-κB) signaling pathway activation were determined by western blot.ResultTet ameliorated the damage to organ tissues, improved coagulation indexes, and reduced the inflammatory cytokine production in LPS-induced mouse DIC. Tet also inhibited TNF-α expression by suppressing NF-κB signaling pathway activation in an in vitro LPS model using RAW 264.7 macrophages.ConclusionsTet has a mitigating and therapeutic effect on the LPS-induced DIC model via anticoagulant and anti-inflammatory effects, showing its potential as an adjunct to DIC treatment.

Bacterial Endotoxin Activates the Coagulation Cascade through Gasdermin D-Dependent Phosphatidylserine Exposure

Excessive activation of the coagulation system leads to life-threatening disseminated intravascularcoagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. Wefound that caspase-11, a cytosolic LPS receptor,activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores andsubsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD poresmediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, orneutralization of phosphatidylserine orTF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α andIL-1β, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.

Bacterial Endotoxin Activates Coagulation Cascades Through GSDMD-Dependent Phosphatidylserine Exposure

Excessive activation of the coagulation system by endotoxin (lipopolysaccharide: LPS), the major cell-wall component of Gram-negative bacteria, leads to life-threatening disseminated intravascular coagulation (DIC). However, the underlying mechanisms remain largely unknown. Here, we show that caspase-11, a cytosolic LPS receptor, activates the coagulation cascade through gasdermin D (GSDMD) and tissue factor (TF), an initiator of coagulation cascades. Upon activation by caspase-11, GSDMD-mediated phosphatidylserine exposure markedly enhances the activation of TF. Genetic deletion or pharmacological inhibition of caspase-11, ablation of GSDMD or neutralization of phosphatidylserine or TF uniformly prevents LPS-induced DIC. Further, plasma concentrations of interleukin (IL)-1α and IL-1β, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores in septic patients. These findings not only identify molecular links between innate immunity and coagulation, but also could open a new avenue to prevent infection-induced DIC.

BMSCs pre-treatment ameliorates inflammation-related tissue destruction in LPS-induced rat DIC model

This study aimed to investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on disseminated intravascular coagulation (DIC) model rats and to further explore the underlying mechanism. A rat model of lipopolysaccharide (LPS)-induced DIC was successfully established, as indicated by impaired plasma hemostatic parameters and damaged organ functions in rats. Importantly, pre-treatment with rat allogeneic BMSCs before LPS injection significantly alleviated systemic intravascular coagulation, reduced plasma levels of organ dysfunction indicators and pro-inflammatory cytokines, suppressed fibrin microthrombi formation, ameliorated liver, heart, and renal injuries, and increased 24-hour survival rates in LPS-induced DIC rats. The protection of BMSCs against DIC was in a moderately dose-dependent manner. Further investigation revealed that BMSCs co-cultured with peripheral blood mononuclear cells (PBMCs) significantly inhibited the LPS-stimulated PBMCs proliferation and the release of pro-inflammatory cytokines from PBMCs. Of note, upregulation of immunosuppressive factors including indoleamine 2,3-dioxygenase and interleukin-10, which was induced by interferon-γ, contributed to BMSCs-mediated inhibition of LPS-stimulated PBMCs proliferation. These effects do not depend on the direct cell–cell contact. In conclusion, BMSCs pre-treatment ameliorates inflammation-related tissue destruction in LPS-induced DIC model rats. The protection of BMSCs may be attributed to their anti-inflammatory and immunomodulatory properties, which render BMSCs a promising source for stem cell-based therapeutic approaches in inflammation-related DIC.

Genistein has the function of alleviating and treating disseminated intravascular coagulation caused by lipopolysaccharide.

Symptoms of disseminated intravascular coagulation (DIC) include thromboembolism, acute attrition bleeding and multiple organ failure. Genistein isolated from leguminous plants has been shown to be effective in oxidation resistance and tumor inhibition. The present study was designed to evaluate the therapeutic effects of genistein in DIC and preliminarily discuss the mechanisms regarding the anti-inflammatory and anticoagulant effect of genistein. Swiss mice were randomly divided into the following groups-(1) lipopolysaccharide (LPS), (2) genistein, (3) dimethyl sulfoxide (DMSO, the non-major solvent component of genistein), (4) DMSO + LPS, (5) saline control group, and (6) heparin control group. LPS was injected intraperitoneally in all the groups except the DMSO group and saline control group. Our results significantly showed that the morphological structure of the liver and kidneys was improved and the fiber protein deposition was decreased, with remarkable improvement of coagulation indicators, function indicators and inflammatory factors in the genistein treatment group compared with the LPS group. In vitro phosphorylated-nuclear factor kappa-light-chain-enhancer of activated B cells and interleukin-6 were obviously reduced in the genistein treatment group compared with the LPS group in RAW 264.7 murine macrophage cells. All the results suggested that genistein has the function of alleviating and treating LPS-induced DIC by anti-inflammatory and anticoagulation effects. We tentatively propose that genistein is a potential drug for auxiliary treatment of DIC.

Abstract 039: A Small Fragment Derived from von Willebrand Factor Improves Survival in a Mouse Model of Endotoxemia-Induced Disseminated Intravascular Coagulation

Introduction: Disseminated intravascular coagulation (DIC) is a life-threatening condition that results in wide-spread clot formation and organ failure. Sepsis-induced DIC is associated with a high mortality rate, prompting the need for an effective therapeutic intervention. Our lab has identified the A2 domain of the von Willebrand factor (VWF) protein as a potent treatment for sepsis-induced DIC. Historically, VWF has only been described as a contributor of clot formation; however, our lab has demonstrated that purified recombinant full length A2 domain of the VWF protein can dampen fibrin-rich microthrombi formation in mice with endotoxin. The mode of action appears to be via the interaction with the cell-surface expressed vimentin on endothelium, inhibiting VWF strings formation and thus, platelet adhesion. Goal: Since the VWF protein is naturally cleaved in circulation by the enzyme ADAMTS-13 via the A2 domain, one can argue whether the beneficial effect observed with full len gth A2 domain is terminated by the cleavage of ADAMTS-13. We produced recombinant proteins encompassing the sequences of the two proteolytic products of the A2 domain; the A2Nt and A2Ct proteins. I will identify which fragment retains the mode of action of the full length A2 domain in a mouse model of endotoxin-induced DIC. Methods: The recombinant A2 and vimentin proteins were produced using the bacterial expression system. Protein: protein interactions were determined by optical interferometry. The endotoxin-induced DIC model was achieved by injecting C57B/6 mice with lipopolysaccharide (LPS). Treatment with A2 or its fragments was started approximately 2 hours after LPS insult. Results: The A2N t protein has a higher affinity for vimentin than the A2Ct. Treatment of A2Nt in a LPS-induced DIC mouse model was effective in maintaining survival in males, a comparable finding that mimics treatment of mice with full-length recombinant A2. Interestingly, A2Nt showed less survival than full length A2 in females. These findings highlight the potential differences in therapy responses between genders, an occurrence that some studies have described to also occur in humans.

Describing the molecular mechanism by which the A2 protein improves survival in mice with endotoxemia.

Von Willebrand factor (VWF) is an essential protein that mediates the platelet adhesion to the exposed subendothelium surface and significantly contributes to arterial thrombosis and stroke. Secreted VWF from the stimulated endothelium as long strings remain anchored to the surface of the endothelial cells and interact with circulating platelets, leading to vessel occlusion. We have shown that the isolated A2 domain of VWF (A2 protein) contains bind activity for the A1 domain, vimentin, and fibrin(ogen). In fact, as a result of those interactions, the A2 protein impairs platelet adhesion to VWF and fibrin(ogen), formation of VWF strings on the endothelium, and fibrin polymerization. The A2 protein was effective in preventing fibrin‐rich microvascular thrombosis and improving survival (100%) of mice with endotoxemia (LPS)‐induced disseminated intravascular coagulation (DIC). Thus, a goal was to identify the binding sites of A1 domain, vimentin, and fibrin in the A2 protein and dissect the mode of action by which the A2 protein recuperates the sick animals. To determine the amino acid residues that are important in the recognition for vimentin, A1 and fibrin, I used the crystal structure of the A2 domain as a guide for point mutations. Two mutations, E1523A and E1567A were introduced in the A2 protein. The purified mutants were tested for their binding capacity for the ligands using ELISA. Moreover, the A2 mutants were tested in mice with endotoxemia (LPS). We are now demonstrating that E1523A mutation had a reduced binding affinity for Vimentin and A1 domain. Strikingly, E1567A and E1523A mutations significantly abolished the binding to fibrin(ogen). Importantly, both mutations failed to replicate the beneficial effect of the wild type A2 protein in improving survival of mice with LPS‐induced DIC. Together, these findings suggest that the A2 protein impairs microvascular thrombosis in mice with endotoxemia mainly via its interaction with fibrin.Support or Funding InformationThis research was supported by funding provided by the following NIH Diversity Supplement: R01GM112806‐0351.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myricetin protects against lipopolysaccharide-induced disseminated intravascular coagulation by anti-inflammatory and anticoagulation effect

Objective: To explore the therapeutic effect and mechanism of myricetin on disseminated intravascular coagulation (DIC). Methods: The DIC model was established by injection of 60 mg/kg LPS in KM mice, and the treatment groups were injected myricetin with different concentrations (25 or 50 mg/kg) 30 min before the model was established. Both coagulation indicators and organ function were tested, including PT, APTT, fibrinogen, AST, ALT, BUN and tissue section. In vitro, the inflammatory model of RAW 264.7 macrophage cells were established by 10 μg/mL LPS. The treatment group was treated with 50 μmol/mL myricetin for 30 min before LPS, and the expression of TNF-α and p-NF-κB was detected, further to explore the therapeutic mechanism. Results: LPS-induced DIC led to a reduction of fibrinogen and a rise of PT, APTT, AST, ALT, BUN levels, but the treatment of myricetin significantly inhibited these abnormalities. Histopathology analysis also revealed that myricetin remarkably protected the liver and renal damage. In vitro, the expression of TNF-α and p-NF-κB induced by LPS was repressed by myricetin. Conclusions: This study provides new insights into the protective effects of myricetin in LPS-induced DIC by anticoagulant and anti-inflammatory via suppressing the activation of p-NF-κB which decreased TNF-α level.

Atractylenolide Ⅰ protects against lipopolysaccharide-induced disseminated intravascular coagulation by anti-inflammatory and anticoagulation effect.

To investigate whether atractylenolide Ⅰ (ATL-Ⅰ) has protective effect on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) invivo and invitro, and explore whether NF-κB signaling pathway is involved in ATL-Ⅰ treatment. New Zealand white rabbits were injected with LPS through marginal ear vein over a period of 6hat a rate of 600μg/kg (10mL/h). Similarly, in the treatment groups, 1.0, 2.0, or 5.0mg/kg ATL-Ⅰ were given. Both survival rate and organ function were tested, including the level of alanine aminotransferase (ALT), blood urine nitrogen (BUN), and TNF-α were examined by ELISA. Also hemostatic and fibrinolytic parameters in serum were measured. RAW 264.7 macrophage cells were administered with control, LPS, LPS+ATL-Ⅰ and ATL-Ⅰ alone, and TNF-α, phosphorylation (P)-IκBα, phosphorylation (P)-NF-κB (P65) and NF-κB (P65) were determined by Western blot. The administration of LPS resulted in 73.3% mortality rate, and the increase of serum TNF-α, BUN and ALT levels. When ATL-Ⅰ treatment significantly increased the survival rate of LPS-induced DIC model, also improved the function of blood coagulation. And protein analysis indicated that ATL-I remarkably protected liver and renal as decreasing TNF-α expression. Invitro, ATL-I obviously decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65), with the decrease of P-IκBα. ATL-Ⅰ has protective effect on LPS-induced DIC, which can elevate the survival rate, reduce organ damage, improve the function of blood coagulation and suppress TNF-α expression by inhibiting the activation of NF-κB signaling pathway.

Bone Marrow-Derived Mesenchymal Stem Cells-Mediated Protection Against Organ Dysfunction in Disseminated Intravascular Coagulation Is Associated With Peripheral Immune Responses.

Disseminated intravascular coagulation (DIC) is a fatal thrombohemorrhagic disorder. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stem cells that have tremendous therapeutic effect. Our aim was to explore whether the immune mechanisms were associated with BMSCs-afforded protection against DIC. We generated a rat model of DIC by lipopolysaccharide (LPS, 3 mg/kg) injection via the tail vein. In the treatment group, rats were pre-treated with 1 × l03 , 1 × l04 , 1 × l05 , and 1 × l06 allogeneic BMSCs before LPS injection. Blood sample was withdrawn from the abdominal aorta at 0 (before), 4, and 8 h after LPS injection and used for biochemical analyses. After experiments, the mice were sacrificed and their organs were harvested and observed by H&E and PTAH staining. Continuous infusion of LPS into the rats gradually impaired the hemostatic parameters and damaged organ functions. However, pre-treatment with BMSCs dose-dependently improved the hemostatic parameters. Meanwhile, the treatment significantly suppressed the fibrin microthrombi formation and alleviated liver, heart, lung, and renal injuries. Flow cytometry analysis demonstrated that BMSCs pre-treatment inhibited LPS-induced upregulation of CD3+ CD8+ T cells and CD3+ /CD161a+ NKT cells in the peripheral blood. BMSCs pre-treatment reversed the upregualtion of the B-cell population and the percentage of CD43+ /CD172a+ monocytes in the DIC models. Finally, BMSCs pre-treatment decreased the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), and interleukin-6 (IL-6) and increased the levels of interleukin-10 (IL-10) in LPS-induced DIC models. Pre-treatment with BMSCs can reduce coagulation and alleviate organ dysfunction via peripheral immune responses in LPS-induced DIC rat model. J. Cell. Biochem. 118: 3184-3192, 2017. © 2017 Wiley Periodicals, Inc.

A novel fibrinogenase from Agkistrodon acutus venom protects against LPS-induced endotoxemia via regulating NF-κB pathway

Context: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines.Objective: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process.Methods: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30 mg/kg LPS, (b) LPS + 0.3 mg/kg FII, (c) LPS + 1.0 mg/kg FII, (d) LPS + 3.0 mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS + FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot.Results: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65).Conclusions: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.

MiR-96 and miR-330 overexpressed and targeted AQP5 in lipopolysaccharide-induced rat lung damage of disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a severe clinical condition that can lead to or aggravate the development of multiple organ dysfunction syndrome. Of all types of organ damage, lung damage is the most frequent and most severe. In DIC patients, lung damage is primarily characterized by pulmonary edema. Aquaporin (AQP) 5 is the chief AQP in the lungs and it plays a key role in many processes, including water transport in normal and abnormal lungs. Here we demonstrate that expression of AQP5 and two microRNAs, miR-96 and miR-330, in rat lung of lipopolysaccharide (LPS)-induced DIC. We also show that both miR-96 and miR-330 can regulate the expression of AQP5 by binding with its 3'-untranslated region (UTR) by luciferase activity assay. These results suggest that microRNAs are involved in lung damage in LPS-induced rat DIC and can be a potential target for molecular therapy.

Antagonism by salvianolic acid B of lipopolysaccharide-induced disseminated intravascular coagulation in rabbits.

The aim of the present study was to investigate the effects of salvianolic acid B on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. Continuous infusion of LPS was used to induce a DIC model in rabbits. Treatment with salvianolic acid B (1, 3 or 6 mg/kg) was started simultaneously with LPS infusion (0.5 mg/kg LPS in 60 mL saline; 10 mL/h over a period of 6 h) through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were determined, as were plasma levels of fibrin-fibrinogen degradation products (FDP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), protein C activity, antithrombin III (ATIII) and tumour necrosis factor (TNF)-α concentration. The gradual impairment of haemostatic parameters was induced by continuous infusion of LPS. There were marked increases in APTT, PT, BUN, ALT and plasma TNF-α and marked decreases in the platelet count, fibrinogen, FDP, protein C and ATIII. The intravenous administration of 1, 3 or 6 mg/kg salvianolic acid B attenuated the increases in APTT, PT, BUN, ALT and plasma TNF-α and the decreases in fibrinogen, platelet, FDP, protein C and ATIII induced by LPS infusion. These observations indicate that salvianolic acid B has an effect against LPS-induced DIC in rabbits.

Effects of quercetin on LPS-induced disseminated intravascular coagulation (DIC) in rabbits

IntroductionQuercetin is widely distributed in plants and has been reported to have effects of anti-inflammation and anti-thrombosis. In this study, we evaluated the protective effect of quercetin on LPS-induced experimental DIC in rabbits, and tried to clarify its mechanism against DIC. Materials and MethodsLPS-induced DIC model in rabbits was established through continuous infusion of 100 ug·kg-1·h-1 LPS for a period of 6h. Six groups were divided: quercetin-treated groups (0.5, 1.0, and 2.0mg·kg-1·h-1, respectively), LPS-control group, heparin-control group (100IU·kg-1·h-1), and saline-control group. APTT, PT, and plasma FIB level were measured, the plasma levels of ALT, BUN, and TNF-α were detected, and the activity of Protein C and ATIII was recorded. ResultsA continuous injection of LPS induced a gradual impairment of hemostatic parameters, a rise in plasma level of TNF-α, and damage in renal and hepatic function. The intravenous administration of quercetin significantly attenuated the increase of APTT, PT, ALT, BUN, and TNF-α, and the decrease of plasma FIB level and activity of Protein C and ATIII. ConclusionQuercetin may have a protective effect against LPS-induced DIC in rabbits through anti-inflammation and anticoagulation.

A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C

The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC

Introduction Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by the widespread activation of coagulation. This leads to failure of multiple organs in the body and finally death. Because there is no effective therapy for DIC, the clinical prognosis is poor and the mortality is high. Materials and Methods The animals were intravenously injected with Lipopolysaccharide (LPS) for 6 hours and simultaneously infected three doses of recombinant fibrinogenase II (rFII) for 2 hour. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelets count, fibrinogen and fibrin-fibrinogen degradation products (FDP) were determined. The plasma levels of alanine aminotransferase (ALT), creatinine (Cr) and tumor necrosis factor-α (TNF-α) were detected. Liver and kidney samples were stained with hematoxylin-eosin and kidney sections were stained with phosphotungstic acid-hematoxylin. Results We observed that rFII increased survival rate in LPS-induced DIC rabbits as well as heparin did. Administration of rFII as well as heparin attenuated the increased plasma levels of APTT, PT and FDP and the decreased plasma level of fibrinogen at 6 h. rFII reduced hepatic and renal damages and decreased the levels of ALT and Cr as well as heparin did. rFII also significantly reduced the increased plasma levels of TNF-α. rFII significantly reduced the kidney fibrin deposits with respect to LPS treated animals. Conclusions Our findings suggest that rFII from Agkistrodon acutus venom could have protective effect on DIC via reducing liver and renal damages and direct degradation of microthrombi.